1. Field of the Invention
This invention resides in the fields of enzymatic reactions and assays for monitoring or detecting enzyme activity. In particular, this invention relates to reporter species that serve as indicators of enzyme activity.
2. Description of the Prior Art
The study and detection of enzyme activity serve a wide range of purposes both in research laboratories and in clinical assays. Enzyme activity is monitored, for example, in monitoring physiological functions in patients during routine checkups or diagnostic procedures in general, in monitoring the exposure of workers and residents to potentially harmful chemicals such as toxic or carcinogenic pesticides or inorganic materials in the atmosphere, soil, or drinking water, in determining the effectiveness of pharmaceuticals on disease states or conditions, in screening new compounds for biological activity as either promoters or inhibitors of particular enzymes, in monitoring gene and transgene expression, and in performing immunological and other laboratory assays such as enzyme-linked immunosorbent assays (ELISAs) and Western blots.
Optical methods of detection, such as fluorescence emission, UV absorptivity, and colorimetry are convenient and highly effective for detecting, monitoring, and measuring enzyme activity, since methods such as these can generate either qualitative or quantitative information and detection can be achieved either by direct visual observation or by instrumentation. Optically detectable reporters, i.e., synthetic or substitute substrates that are added to a sample and that display a measurable increase or other difference in optical detectability upon action of the enzyme, are therefore particularly useful. Examples of optical reporters that are currently known are 4-nitrophenol, xcex1-naphthol, xcex2-naphthol, resorufin and substituted resorufins, nitranilide, ethanol, 5-bromo-4-chloro-3-indole, and umbelliferone derivatives. The degree of change and hence the effectiveness of optical detection reporters depend on any of several factors, depending on the detection method for which they are used. Some of these factors are a high extinction coefficient for reporters that are detectable by light absorptivity (particularly a large increase from substrate to product). a large change in the wavelength at which maximum absorptivity occurs (particularly a large substrate-to-product red shift), a substrate-to-product increase in the Stokes"" shift for fluorescent reporters, and the chemical stability of the reporter.
It has now been discovered that ethers and esters of cyanohydrins are highly effective optical detection reporters for certain types of enzyme activity, notably those that convert the ethers or esters to the cyanohydrins. The cyanohydrins function as proaldehydes and proketones, spontaneously converting to aldehydes and ketones, respectively, under appropriate pH conditions in the neutral to basic range. The use of these cyanohydrin ethers and esters as optical detection reporters derives in each case from a group bonded to the central carbon of the cyanohydrin moiety, the group imparting to the final aldehyde or ketone the optical detectability that serves as the means for detecting the enzyme activity. The effectiveness of these cyanohydrin ethers and esters as reporters derives from large differences in optical properties between the aldehydes and ketones and either the cyanohydrins or the cyanohydrin ethers or esters (i.e., the unconverted reporters). These differences include a larger Stokes"" shift (which in fluorescent reporters is the difference between the maximum excitation wavelength and the maximum emission wavelength) of the product (aldehyde or ketone) relative to the substrate (cyanohydrin ether or ester), a larger substrate-to-product red shift (the difference in maximum absorbance in UV and calorimetric assays and the difference in emission maximum in fluorescence assays), a larger quantum yield (in fluorescence assays the proportion of energy absorbed that is then emitted as light), and a greater extinction coefficient at the maximum wavelength. These differences provide increases in sensitivity and decreases in background relative to prior art reporters, permitting the use of a wider band width, simpler and less costly optics, and various other benefits to the assay as a whole.
This invention resides in novel cyanohydrin esters and ethers as well as novel methods for detection of enzyme activity involving the use of cyanohydrin esters and ethers, both novel and already known, as reporter substances. The many applications and methods of implementation of these substances will be better understood from the description that follows, and further applications and uses will be readily apparent to those skilled in the art.